Calcium requirement estimated by balance study in elderly Japanese people.

The recommended dietary allowance (RDA) for calcium (Ca) of Japanese adults is proposed to be 600 mg/day, which is lower than those of other countries. In this report we estimated the Ca requirement and the RDA for Ca in elderly Japanese utilizing a Ca balance method. Subjects were 10 men aged 65-72 years and 10 women aged 62-77 years. Following a 14 day adaptation period, each participant was subjected to a low Ca diet (Ca 250 mg as a meal) for 6 days. After an interval of 2 weeks or more, another 14 day adaptation period was set and then a high Ca diet (Ca 250 mg as a meal and 600 mg as CaCO3) was served to the subjects for 6 days. Ca balance was calculated at each dose of Ca intake. Ca requirement was estimated by the intersection of the average Ca intake-retention diagram. Daily Ca requirement was 702 mg in the men and 788 mg in the women. The Ca requirement values were multiplied by 1.2 to obtain the RDA for Ca. As a result, RDA for Ca was 842 mg/day for men and 946 mg/day for women. When these values were normalized with the body weight, the RDA for Ca of Japanese and Caucasian women was similar (18.1 and 18.5 mg/kg body weight per day, respectively). Our results suggest the difference in Ca balance between the genders and among populations may be ascribed at least partly to differences in body size. In addition, body weight should be considered when comparing the RDAs among different populations.

Change in calcium balance and bone mineral density during pregnancy in female rats.

This study investigated changes in Ca balance and BMD during pregnancy in female rats. During pregnancy, the intestinal Ca absorption increased significantly, and Ca accumulation was also markedly elevated. However, BMD values for the lumbar spine decreased significantly during pregnancy. Twenty female SD rats, 10 weeks of age (Japan SLC Co., Shizuoka, Japan) were acclimated for 2 weeks. Then, the rats were divided into two groups; the control (no pregnancy) group (n=10) and the pregnant group (n=10). The rats in the pregnant group were kept in a cage with a male rat for 5 days at 12 weeks of age, and all 10 were successfully impregnated. During the pregnancy period, the values for intestinal Ca absorption and the rate of the intestinal Ca absorption in the pregnant group were significantly greater than those of the control group. In addition, in spite of the significant increase in urinary Ca excretion in the pregnant group, the Ca accumulation was markedly elevated during the latter half of pregnancy. On the other hand, the BMD value in the lumbar spine for the pregnant group significantly decreased during pregnancy. These findings suggest that pregnancy accelerated intestinal Ca absorption and Ca accumulation in female rats, while the lumbar spine BMD decreased during pregnancy.

Effect of 1alpha-hydroxyvitamin D3 and egg-shell calcium on bone metabolism in ovariectomized osteoporotic model rats.

Egg-shell calcium (Ca) is one of the effective Ca sources for bone metabolism. In the present study, we investigated whether egg-shell Ca had similar effects compared with calcium carbonate (CaCO3) when vitamin D3 (1alpha(OH)D3) treatment was given to an osteoporotic rat model. In both 1alpha(OH)D3-supplemented and -unsupplemented rats, the bone mineral density (BMD) of the lumber spine in the vitamin-supplemented group increased significantly compared with the unsupplemented group. In a Ca balance study, there were also significant differences in intestinal Ca absorption, urinary Ca and fecal Ca between the vitamin-supplemented and -unsupplemented groups. These results show that egg-shell Ca could have similar effects to CaCO3 on bone metabolism. In contrast with CaCO3, vitamin D3 supplementation did not significantly increase serum Ca levels in the egg-shell Ca group; however, the mechanism of Ca absorption is still unclear. Our results suggest that egg-shell Ca may be an effective nutrient in Ca metabolism for people treated with vitamin D3.

[Calcium intake and osteoporosis in many countries].

From the viewpoint of preventing the life-style related diseases, daily diet and nutrient intake are important. In particular, an adequate intake of calcium is one of the important means of preventing osteoporosis. Calcium intake is much lower in Asia and Africa than in the United States and Europe, mainly due to the exceedingly low intake of milk and dairy products. However, although the prevalence of osteoporosis, especially hip fracture, is currently much higher in Western countries than in developing Asian countries, in the rapidly industrializing countries of Asia and other areas, the prevalence of osteoporosis is rapidly approaching the level in Western countries. As the world population continues to age, it is necessary to consider the prevention of osteoporosis from the viewpoint of a global problem.

[Phosphorus intake and osteoporosis].

Phosphorus (P) is one of the most important nutrients for bone metabolism, such as calcium. In general, P intake is usually adequate in our daily diet, and there is a risk of over-consumption from processed food. On the other hand, Ca intake is not always adequate from the Japanese daily diet. When Ca/P is taken from the daily diet at a level of 0.5 - 2.0, the P intake level dose not affect intestinal Ca absorption. Therefore, it is important not only to pay attention to preventing the over-consumption of P, but also to obtain a sufficient intake of Ca. For the prevention of osteoporosis, it is important to consume sufficient Ca and to maintain and appropriate Ca/P balance from diet.

Adverse effects of an active fragment of parathyroid hormone on rat hippocampal organotypic cultures.

Adverse effects of an active fragment of parathyroid hormone (PTH(1 - 34)), a blood Ca(2+) level-regulating hormone, were examined using rat hippocampal slices in organotypic culture. Exposure of cultured slice preparations to 0.1 microM PTH(1 - 34) for 60 min resulted in a gradual increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)); this effect was most obvious in the apical dendritic region of CA1 subfield. When PTH(1 - 34) at a lower concentration (1 nM) was added to the culture medium and its toxic effects examined using a propidium iodide intercalation method, significant toxicity was seen 3 days after exposure and increased with time. Cells in the CA1 region seemed more vulnerable to the hormone than cells in other regions. At 1 week of exposure, the toxic effects were dose-dependent over the range of 0.1 pM to 0.1 microM, the minimum effective dose being 10 pM. The adverse effects were not induced either by the inactive fragment, PTH(39 - 84), or by an active fragment of PTH-related peptide (PTHrP(1 - 34)), an intrinsic ligand of the brain PTH receptor. The PTH(1 - 34)-induced adverse effects were significantly inhibited by co-administration of 10 microM nifedipine, an L-type Ca(2+) channel blocker, but not by co-administration of blockers of the other types of Ca(2+) channel. The present study demonstrates that sustained high levels of PTH in the brain might cause degeneration of specific brain regions due to Ca(2+) overloading via activation of dihydropyridine-sensitive Ca(2+) channels, and suggests that PTH may be a risk factor for senile dementia. British Journal of Pharmacology (2000) 129, 21 - 28

Phosphate depletion enhances tissue-nonspecific alkaline phosphatase gene expression in a cultured mouse marrow stromal cell line ST2.

Alkaline phosphatases (ALP) are highly ubiquitous enzymes present in the majority of animals from bacteria to higher vertebrate. Although their wide distribution in nature has suggested that these enzymes should perform important biological functions, their detailed roles or natural substrates remain unknown. In Escherichia coli, the extracellular phosphate (Pi) limitation induces the ALP gene, indicating the role of extracellular Pi in ALP gene regulation. However, little is known about the similar mechanisms in mammalian cells. This study was designed to examine the effect of low Pi medium on the ALP activity and its expression in the mouse stromal cell line ST2. The enzymatic property was classified into tissue-nonspecific ALP (TNSALP). After treatment by Pi starvation for 3 days, there was a 2-fold increase in the specific activity of TNSALP. RT-PCR analysis revealed that the mRNA of the TNSALP gene was highly stimulated. These results indicated that the effect of Pi depletion on ALP activity was regulated at the TNSALP transcriptional level, suggesting that the possible role of the Pi sensing system for biological functions of ALP might have been conserved in evolution. Our findings also made it possible to discuss the physiological roles of ALP in vivo.

The advantage of alfacalcidol over vitamin D in the treatment of osteoporosis.

Although alfacalcidol is widely used in the treatment of osteoporosis, its mechanism of action in bone is not fully understood. Alfacalcidol stimulates intestinal calcium (Ca) absorption, increases urinary Ca excretion and serum Ca levels, and suppresses parathyroid hormone (PTH) secretion. It remains to be clarified, especially under vitamin D-replete conditions, whether alfacalcidol exerts skeletal effects solely via these Ca-related effects, whether the resultant suppression of PTH is a prerequisite for the skeletal actions of alfacalcidol, and, by inference, whether alfacalcidol has an advantage over vitamin D in the treatment of osteoporosis. To address these issues, we (1) compared the effects of alfacalcidol p.o. (0.025-0.1 microg/kg BW) vis-à-vis vitamin D(3) (50-400 microg/kg BW) on bone loss in 8-month-old, ovariectomized (OVX) rats as a function of their Ca-related effects, and (2) examined whether the skeletal effects of alfacalcidol occur independently of suppression of PTH, using parathyroidectomized (PTX) rats continuously infused with hPTH(1-34). The results indicate that (1) in OVX rats, alfacalcidol increases BMD and bone strength more effectively than vitamin D(3) at given urinary and serum Ca levels: larger doses of vitamin D(3) are required to produce a similar BMD-increasing effect, in the face of hypercalcemia and compromised bone quality; (2) at doses that maintain serum Ca below 10 mg/dl, alfacalcidol suppresses urinary deoxypyridinoline excretion more effectively than vitamin D(3); and (3) alfacalcidol is capable of increasing bone mass in PTX rats with continuous infusion of PTH, and therefore acts independently of PTH levels. It is suggested that alfacalcidol exerts bone-protective effects independently of its Ca-related effects, and is in this respect superior to vitamin D(3), and that the skeletal actions of alfacalcidol take place, at least in part, independently of suppression of PTH. Together, these results provide a rationale for the clinical utility of alfacalcidol and its advantage over vitamin D(3) in the treatment of osteoporosis.

[Relation between calcaneal bone mass and lifestyles in young adult women. Influence of experience of delivery and months post delivery].

The aim of this study was to clarify the cross-sectional relation between calcaneal bone mass and lifestyles in healthy pre-menopausal young adult women classified by the experience of pregnancy and the months after delivery. The 457 healthy Japanese women aged 20-39 years living in an urban community underwent a health check up for osteoporosis. Calcaneal bone was measured by ultrasound using the Lunar Achilles, and stiffness was used as an index of bone mass. The information on pregnancy, delivery and lactation, and lifestyles including past and current exercise and frequency of food consumption were collected by a questionnaire. In addition, two-day dietary records were obtained to assess the nutrient intake, and seven-day walking records as an index of physical activities. These subjects were divided into 3 groups according to the experience of pregnancy, and the time after delivery (subgroups of no experience of pregnancy, 12-35 months post-delivery and 36 months or more post-delivery), and the relation between stiffness index and lifestyle factors was examined in the 3 groups. 1) Stiffness correlated significantly with experience of pregnancy, age at menarche, current exercise and frequency of intake of dairy products. 2) In the group without experience of pregnancy, stiffness in women with current exercise habit was significantly higher than women without the habit. Those with past exercise habit, current milk consumption and current calcium intake showed relatively greater stiffness. 3) In the 12-35 months post-delivery group, there was no relation of past and current physical activity to stiffness. For women whose frequency of milk consumption or daily calcium intake were high, the stiffness was significantly greater. In addition, past milk consumption and current frequency of dairy products consumption tended to show higher stiffness. 4) In 36 months or more post-delivery group, women with current exercise habit exhibited significantly higher stiffness than women without the habit. Low calcium intake tended to show low stiffness. These results indicated that the effects of lifestyle on calcaneal bone mass were different depending on the experience of pregnancy and the months after delivery in pre-menopausal young adult women.

Activation of dihydropyridine sensitive Ca2+ channels in rat hippocampal neurons in culture by parathyroid hormone.

We examined the effects of parathyroid hormone (PTH) on rat hippocampal neurons in culture to determine whether it caused a similar intracellular calcium concentration ([Ca2+]i) increase in these cells to that seen with renal epithelial cells and found that PTH induced the effect in about 30% of the neurons. The effects appeared gradually during continuous administration of full-length PTH(1-84) or its active fragment, PTH(1-34), but not of an inactive fragment, PTH(39-84). However, the active fragment of the PTH-related peptide (PTHrP(1-34)) had little effect on [Ca2+]i during 60 min of administration. The PTH effect was inhibited by nifedipine, an L-type Ca2+ channel antagonist, and facilitated by S-(-)-BAY K 8644, an L-type Ca2+ channel agonist. Our findings suggest that PTH is one of the causal factors for the age-related increase in the density of voltage gated Ca2+ channels in hippocampal neurons.

The maltitol-induced increase in intestinal calcium transport increases the calcium content and breaking force of femoral bone in weanling rats.

Maltitol is a disaccharide alcohol that is produced by hydrogenation of maltose and exhibits resistance to intestinal disaccharidases. We demonstrated previously that maltitol stimulated transepithelial diffusional calcium transfer in the ileum, accompanied by an elevation of intestinal calcium absorption as well as calcium retention in the body. In this study we examined whether the maltitol-induced increase in the diffusional transfer of intestinal calcium absorption leads to an alteration of the physical properties of bones in the weanling rats which exhibit the maximal level of intestinal active calcium absorption. Rat pups were removed from dams at 24 d of age and were fed the diets containing either maltose (control) or maltitol and a requisite amount of calcium (0.52%) for 21 d. Balance studies performed during the final 5-d period showed that maltitol-fed rats had greater calcium retention and calcium absorption. The breaking force of femoral bones was 13% greater in the rats fed the maltitol diet than in controls. The calcium content and dry weight of both femurs and tibias, as well as the bone mineral density of tibias, were elevated in the rats fed the maltitol diet. In a separate experiment, gastric intubation of maltitol-containing diet increased the serum calcium concentration in the portal vein at 2 and 4 h compared to controls. These results indicate that the maltitol-induced increase in the intestinal calcium absorption through paracellular pathway leads to enhancement of the calcium content and the breaking strength in the bone of weanling rats.

Modulation of bone mass and turnover in growing rats by voluntary weight-bearing exercise and glucose supplementation.

Female Sprague-Dawley rats, 9 weeks of age, were assigned to four groups: Group 0 (n = 8) was dissected for base-line control, and the other three groups were fed for 3 mo: Group 1 (n = 9), sedentary controls; Group 2 (n = 6), running rats housed in a cage with a treadmill and pair-fed with Group 1; and Group 3 (n = 7), running rats, pair-fed and allowed free access to additional glucose. The distances of voluntary running did not significantly differ between Groups 2 and 3. Menstrual cycles in these rats were apparently maintained as observed from daily running distances. The amount of glucose taken by rats in Group 3 was 3.5 +/- 0.4 (mean and SE) g/d. Body weight (BW) at the end of the experiment for Groups 1, 2, and 3 were 295.0 +/- 7.9, 211.7 +/- 5.4 (p < 0.001 vs. Group 1), and 259.0 +/- 3.5 g (p < 0.01 vs. Group 2), respectively. The parameters of bone mass such as ash weights of the femur and bone mineral content of the lumbar spine and the tibia in Groups 1 and 2 did not differ, but the values were significantly greater in Group 3 than in Group 2. However, these parameter values corrected for BW were significantly greater in Group 2 than in Group 1 and did not significantly differ between Groups 2 and 3. The parameters of bone formation, such as serum bone alkaline phosphatase activity levels and trabecular bone formation rates corrected for BW, were significantly greater in Group 2 than in Group 1 but did not differ between Group 2 and 3. However, the parameters of bone resorption, such as serum tartrate resistant acid-phosphatase levels, were significantly less in Group 3 than in Group 2. These results suggest that voluntary running augments the age-dependent increase in bone mass by modulating the bone turnover when an adequate energy source is supplied under conditions of normal menstruation, and an adequate supply of energy could be necessary to enhance the age-dependent increase in bone mass.

[Nutritional therapy].

Osteoporosis is one of the life style related diseases. Therefore, from the viewpoint of prevention, daily diet and nutrient intake are important. An adequate intake of calcium together with nutrient balance plays an essential role in maintaining and promoting health and preventing osteoporosis. Moreover, prior to nutritional therapy, it is essential to understand the current nutritional status and dietary habits of the individual.

Effect of long-term alcohol administration on bone metabolism in rats.

The purpose of this study was to investigate the effects of different degrees of alcohol ingestion on bone strength and mineral density. Three different groups of growing female rats were administered different doses of an alcohol-water solution for a period of 6 months. These three groups were divided into: 1) the control group, which was only given water; 2) the moderate group, which was given 5% ethanol solution for only 2 h per day; and 3) the excess group, which was given only 5% ethanol solution for 163 days. This ethanol consumption induced no detrimental effect on biochemical parameters including liver function. The moderate group showed significantly higher (p < 0.05) levels of proximal metaphysis as compared to the control group, while there was no difference between the excess group and the control group. Similarly, in comparison to the control group, the moderate group exhibited a significant increase (p < 0.001) in bone mechanical strength, while the excess group showed either the same or decreased bone stiffness. These results indicate that alcohol intake has both beneficial and hindering effects on the skeleton, depending on the concentration and frequency of ethanol intake.

Effects of ovariectomy on intestinal alkaline phosphatase expression in rats.

The aim of this study was to investigate the effects of ovariectomy (OVX) on intestinal alkaline phosphatase (ALP) activity in rats. The calcium (Ca) and phosphorus (P) contents and the mechanical strength of bone were decreased significantly by OVX. Two kinds of mRNAs of rat intestinal ALP (RTIN-1 and RTIN-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). In OVX rats, the level of RTIN-2 mRNA was lowered significantly, while that of RTIN-1 mRNA did not change. This result was compatible with the results of enzymatic activity. This finding suggests the possibility that OVX affects bone metabolism not only directly but also in an indirect way through an intestinal Ca and/or P metabolism via regulation of intestinal RTIN-2 ALP expression.

Effects of physical characteristics and dietary habits on bone mineral density in adolescent girls.

In the health management of bone, of most importance is how to spend the period until peak bone mass, that is appropriate self management for bone health. Therefore, we evaluated the effects of physical characteristics and dietary habits on the bone mineral density (BMD) of the second metacarpal bone (sigma GS/D: BMD) by the digital image processing method (DIP) in 197 healthy adolescent girls (Japanese students at a junior high school, aged 12-15 y), an important period of physical and bone growth. Concerning the physical characteristics of the subjects according to age group, body height in each age group was higher than the standard values for Japanese according to age, but body weights in the 14-year-old and 15-year-old groups were significantly lower than the standard values for Japanese. Compared with the standard BMD values for Japanese according to age, BMD in the subjects was high in the 12-year-old, 13-year-old and 14-year-old groups but low in the 15-year-old group (-7.3%). Concerning the nutritional state, energy, calcium (Ca), and iron intakes were insufficient in every age group. BMD relative to the standard BMD value for Japanese (standard value was regarded as 0%) was evaluated according to the ingestion frequency of Ca-rich foods. The relative BMD value (%) increased with the ingestion frequency of Ca-rich foods. These results suggest that maintenance of an appropriate physique and adequate intake of nutrients such as Ca are important for bone growth during adolescence. Active promotion of educational guidance mainly on the effects of diet on bone health in adolescents in necessary.

Expression of mRNA encoding intestinal type alkaline phosphatase in rat liver and its increase by fat-feeding.

The presence of types of alkaline phosphatase (ALP) other than the tissue non-specific type enzyme in rat liver and its increase by fat feeding are known. In order to examine expression of intestinal type ALP in liver, specific oligonucleotide primers corresponding to two types of mRNAs of rat intestinal ALP (RTIN-1 and -2) were designed and amplified by means of the reverse transcriptase-polymerase chain reaction (RT-PCR). It was found that RTIN-1 mRNA was expressed only in the intestine but not in the liver, while RTIN-2 mRNA was expressed both in the intestine and in the liver. By fat feeding, expression of RTIN-1 mRNA increased in the intestine and that of RTIN-2 mRNA increased both in the intestine and in the liver. Thus, it was concluded that rat liver expressed one of the intestinal type ALP (RTIN-2) which was enhanced by fat feeding.

Ovariectomy decreases osteogenetic activity in rat bone.

The purpose of this study was to investigate the effect of ovariectomy on osteoinductive activity in the bone in rat. Homograft implantation of decalcified humeral diaphysis from ovariectomized or sham-operated rats was performed and harvested after several time periods. A significant decrease in bone induction was found in terms of soft X-ray photography, alkaline phosphatase activity, mineral content and expression of osteocalcin (BGP; bone gla-protein) in the implants from the ovariectomized group in comparison to those from the sham-operated animals. This result suggested that the level of osteoinductive activity, probably due to bone morphogenetic protein, decreased in ovariectomized animals.

Dietary maltitol increases calcium content and breaking force of femoral bone in ovariectomized rats.

Maltitol is a disaccharide alcohol generated by hydrogenation of maltose and exhibiting resistance to intestinal disaccharidases. We demonstrated previously that maltitol stimulates transepithelial transport of calcium in the ileum, accompanied by an elevation of intestinal calcium absorption as well as calcium retention in the body. In this study, we examined whether the maltitol-induced increase in intestinal calcium absorption leads to an alteration of the physical properties of bones in rats subjected to ovariectomy. We used this study as a simulation model for postmenopausal females who are at risk for osteoporosis. Following the intake of a low-calcium diet for 28 d ovariectomized rats were fed diets containing either 10% maltose (control) or 10% maltitol, together with increased amounts of calcium (0.3% in Experiment 1 and 1.2% in Experiment 2) for 21 d. Balance studies performed during the final 5-d (Experiment 1) or 2-d (Experiment 2) period of the experiments showed that maltitol increased intestinal calcium absorption and retention. The breaking force of femoral bones was significantly elevated (by 5-7%) in animals fed the maltitol diet compared with that in rats fed the maltose diet. The calcium content in the femoral bones as well as the mineral bone density of the tibial metaphysis was also elevated in rats fed the maltitol diet. These results indicate that maltitol stimulates the intestinal absorption of dietary calcium leading to an increase in calcium content in the bone, and coinciding with the elevation of the breaking strength of the bone in ovariectomized rats.

The effect of ovariectomy on bone metabolism in rats.

It is well known that the ovariectomized rat is a good model for osteoporosis. Recently, it has been possible to measure the bone mineral density (BMD) of small animals accurately using dual energy X-ray absorptiometry (DXA). Therefore, in this study the change in the BMD in the different bone structures was examined in in vivo using aged and growing rats. The BMD of the lumbar spine and the tibial proximal metaphysis, which are mainly trabecular bone, in ovariectomized aged and growing rats with ovariectomized was significantly lower than those of the control (sham) group. However, the BMD of the tibial diaphysis, which is mainly cortical bone, showed no change nor had a tendency to decrease in the early stages of the experiment. These results suggest that it is important to take into account the age and the bone site in order to evaluate the experiment, which uses the rat model with ovariectomized osteoporosis.